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1- Making Density Gradients PowerPoint PPT Presentation


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1- Making Density Gradients. Pre-formed discontinuous gradients Pre-formed continuous gradients Self-generated gradients. 2- Density Gradient Harvesting. C. A. B. Discontinuous gradient by overlayering. A. B. C. D. Discontinuous gradient by underlayering. density. top. bottom.

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1- Making Density Gradients

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1- Making Density Gradients

  • Pre-formed discontinuous gradients

  • Pre-formed continuous gradients

  • Self-generated gradients

2- Density Gradient Harvesting


C

A

B

Discontinuous gradient by overlayering


A

B

C

D

Discontinuous gradient by underlayering


density

top

bottom

Diffusion of a discontinuous gradient


Discontinuous

Continuous

45-60 min

Rapid formation of continuous from discontinuous gradient


A

B

T

SB

SB

P

M

Two-chamber device for continuous gradients


B

The Gradient Master


Density (g/ml)

2 min

1.13

2.5 min

3.0 min

1.11

1.09

1.07

1

3

5

7

9

Fraction Number

Gradient Master profiles from 10% and 40% iodixanol at 80° and 20 rpm: effect of time


axis of rotation

g

At rest

Spinning

g

Swinging-bucket rotor


Fixed-angle

Vertical

g

1g

Fixed-angle and vertical rotors


Swinging-bucket

Axis of

rotation

Vertical

Sedimentation path length of rotors


A

B

Axis of rotation

Sample/gradient in vertical rotor


time

g

Self-generated gradients

  • At 360,000gav iodixanol molecules will start to sediment


350,000g

350,000g

1g

1g

Self-generated gradient strategy


Near-vertical rotors are best of all


Iodixanol self-generated gradient requirements

  • Rotors with a short sedimentation path length ~ 17 mm

  • RCF of 180,000-350,000gav


Density (g/ml)

15% iodixanol

1.30

1 h

3 h

1.25

20% iodixanol

1.20

1 h

3 h

1.15

1.10

1.05

1.00

1

3

5

7

9

11

13

15

Fraction Number

Effect of time on iodixanol self-generated gradient in the Beckman TLN100 at 365,000g


1.3

15% (170,000g)

30% (170,000g)

15% (353,000g)

1.2

30% (353,000g)

Density (g/ml)

1.1

1.0

1

3

5

7

9

11

Fraction Number

Effect of RCF and [iodixanol] in Beckman VTi65.1 (3 h)


Removal of banded material using a syringe


Collecting a band from a sealed tube


Gilson Fraction

Collector

P

96-well plate

Harvest gradient into equal volume fractions by tube puncture


Perfluorodecalin

Perfluorodecalin

P

P

Upward displacement


P

P

P

P

M

B

EP

C

1

2

3

4

Labconco Auto Densi-flow collects from meniscus


MB

Labconco Auto Densi-flow linked to a Gilson FC205 Fraction Collector


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